Rickettsia cause life-threatening vector-borne diseases, including Anaplasmosis and Ehrlichiosis, which are greatly increasing in worldwide prevalence. There are two major barriers to progress for the development of effective therapies and prophylactic measures for diseases caused by these obligatory intracellular bacteria: 1) the important disease-associated bacterial and host molecules are intracellular, protected from direct antibody (Ab) or drug attack, and 2) bacteria-specific lethal targets are difficult to identify because conventional approaches, such as knockout mutants of essential genes for obligatory intracellular infection, are not feasible. Ehrlichia chaffeensis (Ech) is a classic example. The Type IV Secretion System (T4SS) is conserved among all Rickettsial organisms and is essential for host infection. The recent elucidation of the Ech T4SS effectors Ehrlichial translocated factors 1 and 2 (Etf-1 and Etf-2) provide critical targets for innovative molecular approaches. Nanobodies are the smallest intact antigen-binding fragments (VHH) derived from heavy chain- only antibodies in camelids. Nanobodies are proteolytically stable and biologically active in reduced intracellular environments; therefore, they have greater potential as therapeutic agents in Ech infection and research tools than conventional antibodies. VHH can be cloned into a mammalian expression plasmid, and expressed intracellularly at high levels in heterologous systems. Indeed, we showed transfection of VHH that binds human heterogeneous nuclear ribonucleoprotein K can block Ech infection in human cells. The goal of the proposed research is to carry out a pilot study to develop and characterize Ech infection-blocking intracellular nanobodies for future therapeutic application. We will achieve this goal through two aims. Aim 1 is to isolate and characterize Etf-1 and Etf-2-specific iAbs that inhibit Ech infection by: 1) cloning VHH cDNA from Etf-1 and Etf-2-immunized llama lymphocyte mRNA into a phage display library and isolating Etf-1 and Etf-2 antigen-specific VHHs by panning; 2) cloning Etf-1 and Etf-2-specific VHHs into a mammalian expression vector to produce iAbs by transfection; 3) identifying Ech infection-blocking iAbs and determining their amino acid sequences; and 4) analyzing the mechanism(s) by which the iAbs inhibit Etf-1, -2 functions and block Ech infection. Aim 2 is to deliver selected VHH peptides conjugated with cell penetrating peptide (VHH-CPP) to block Ech infection by: 1) optimizing intracellular delivery of Ech infection-blocking VHH-CPP into human cells in vitro; 2) testing the efficacy of selected VHH-CPP for inhibition of Ech infection in vitro; and 3) analyzing VHH-CPP pharmacokinetics, toxicity, and efficacy for inhibiting Ech infection in mice. Our results will demonstrate the novel use of nanobody technology and CPP to overcome current barriers to advance basic and translational research on obligatory intracellular infection. Eventually, our findings will provide the important scientific basis for a novel anti-rickettsial therapeutic approach.